Tumor Infiltrating Immune Cells in Melanoma | Lenka Vaňková
Immunohistochemical and stereological analysis of immune cell infiltration in metastatic melanoma
Authors: Lenka Vaňková (1), Ondřej Fiala (2), Kristýna Pivovarčíková (3), Samuel Vokurka (2), Martin Pešta (4), Jiří Polívka (1)
Supervisor: Věra Křížková (1)
(1) Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University (2) Department of Oncology and Radiotherapeutics, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (3) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (4) Department of Biology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Melanoma is a highly aggressive skin cancer characterized by significant immune interactions within its tumor microenvironment. Despite significant advances in immunotherapy, the treatment of metastatic melanoma remains challenging. While effectiveness varies among patients, immune checkpoint inhibitors have improved survival rates. Immune cells that infiltrate tumors are essential for the immune response against melanoma. Among these, tumor-infiltrating lymphocytes (TILs) play a crucial role in disease progression and treatment response. Most published studies evaluate only a limited number of parameters, and their results are inconsistent regarding the predictive value of different subtypes of TILs and other immune cells.
Objective: This study aimed to evaluate the infiltration of immunohistochemically positive (IHC+) cells in metastatic melanoma and their potential associations with metastatic site (lymph node vs. skin), immune-related toxic reactions, and different responses to immunotherapy.
Material and Methods: The study included 28 patients diagnosed with metastatic melanoma who received immunotherapy. Various immune cell types infiltrating melanoma metastases were histologically quantified using multilevel sampling and stereological techniques. Immunohistochemical analysis was used to evaluate the expression of the markers CD1a, CD1d, CD3, CD4, CD8, CD20, CD56, CD68, FOXP3, LAG3, PD1, and PD-L1.
Results & Discussion: We demonstrated that the area fraction of all immune cell indicators differed between skin and lymph node metastases. There were variations in CD3 and PD-L1 markers between patients with and without immunotherapy-related toxic effects. In patients with varying responses to immunotherapy, we identified three markers (CD1a, CD20, and PD-L1) that may serve as prognostic indicators. When analyzing a more homogeneous subset of lymph node metastases, CD1a, CD8, and PD-L1 played a similar role.
Conclusion: Our study identified biomarkers that may help predict immunotherapy-related toxicity and treatment efficacy. Tumor-infiltrating immune cells have a significant role in forming the tumor microenvironment and influencing treatment efficacy.
Funding: The grant SVV 260 773 and the Cooperatio program area MED/DIAG and ONCO supported the study.
Study program: Doctoral study – Anatomy, Histology and Embryology | Year of study: 4
ID: 1079
T Cells Impact Prognosis of Colorectar Cancer | Esraa Ali
Prognostic value of T cells in primary colorectal cancer and adjacent normal mucosa in patients with liver metastases
Authors: Esraa Ali (1), Kari Hemminki (1), Andriy Trailin (1)
Supervisors: Kari Hemminki (1), Andriy Trailin (1)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: 25%-50% of early-stage Colorectal cancer (CRC) patients progress to either
synchronous or metachronous metastases, which predominantly affect the liver and drastically
worsen survival. The prognosis for CRC with liver metastasis (LM) remains poor. Prognostic performance of T cells was demonstrated in CRC with LM. However, integral assessment of different T cells subsets was rarely performed in a single cohort. Additionally, creating prognostic models on data available from the primary tumor and the normal colonic mucosa (NM) was less considered. Our hypothesis posits that quantification and comparative analysis of different T cells in and between primary CRC and adjacent NM might have a prognostic value in CRC patients with respect to the presence of synchronous or metachronous LM.
Objective: To determine if analysis of T cell subsets in tumor center of primary colorectal cancer (CRC), adjacent normal mucosa and ratio between those sites can improve prognostic accuracy in CRC patients with the respect to presence of synchronous and metachronous liver metastasis (LM).
Material and Methods: Paired formalin-fixed paraffin-embedded specimens of primary
colorectal cancer and tumor-adjacent normal mucosa (NM) were collected from patients, who
either already had LM (synchronous, n= 55) or developed them thereafter (metachronous, n=44). After immunohistochemical staining, several subsets of T cells (CD3+, CD8+, CD45RO+, CD4+, and Foxp3+) were quantified in NM and tumor center of pCRC. Immune cell densities in NM and TC as well as their ratios (TC/NM) were correlated with clinical and pathological variables and tested as prognostic variables for overall survival (OS).
Results & Discussion: In both synchronous and metachronous groups, NM exhibited higher densities of CD3+, CD8+, CD45RO+, and CD4+ T cells compared to TC. Conversely, Foxp3+ cells were significantly more abundant in TC than in NM. CD45RO+ cells showed positive correlation with both CD8+ and Foxp3+ cells in TC of patients with synchronous LM. Densities of no cell type in the metachronous group were associated with OS. In synchronous group, patients with high densities of Foxp3+ T cells in NM and TC had longer OS in Cox and Kaplan-Meier analyses. Additionally, high ratio of CD45RO+ T cells between TC and NM was associated with longer OS in both types of analyses. The findings support parallel evaluation of immune cells in pCRC and adjacent NM for informed prognostication and warrant further exploration.
Conclusion: Our study revealed significant differences in the T cell landscape between pCRC
and adjacent NM with Foxp3+ cells predominating in TC of pCRC and other cell types predominating
in NM. Favorable prognostic associations of Foxp3+ and CD45RO+ T cells were shown only in synchronous group.
Funding: This research was funded by the grant AZV NU21-03-00506
Study program: Doctoral study – Experimetnal Surgery | Year of study: 4
ID: 1076
Using ATRA to Imporve iNKT Killing | Eliška Jandová
ATR A increases iNKT cell-mediated targeting in hematological malignancies by inducing CD 1d expression
Authors: Eliška Jandová (1), Jana Skálová (1,2), Adéla Makrlíková (1), Robin Klieber (1,2), Pavel Ostašov (1), Daniel Lysák (2), Valentina S Caputo (3), Monika Holubová (1,2)
Supervisor: Monika Holubová (1,2)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (2) Department of Hematology and Oncology, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (3) Cancer Biology and Therapy Laboratory, School of Applied Sciences, London South Bank University, London, UK
State-of-the-Art: Invariant natural killer T cells (iNKTs) are a rare heterogeneous lymphocyte subpopulation that are at the border of innate and adaptive immunity. iNKTs are activated by lipid antigens presented via the CD1d molecule which is expressed on many cell types including tumor cells. During tumor progression, malignant cells tend to decrease levels of CD1d to escape immune surveillance. All-trans retinoid acid (ATRA), a commonly used chemotherapeutic agent, has the ability to increase CD1d expression on the cell surface, making cancer cells more susceptible to iNKT cell-mediated targeting. However, CD1d axis alone is sometimes insufficient without lipid antigen bound. Therefore, the strategies to improve iNKT killing are being investigated.
Objective: Here we investigated the use of ATRA to regulate CD1d expression in various hematological cell lines and monitored its effect on iNKT-mediated cytotoxicity.
Material and Methods: iNKT cells (n=5) were isolated from mononuclear cells and cultured in presence of irradiated (25 Gy) C1R cells engineered to express CD1d, artificial antigen α-galactosylceramide (α-GalCer) (1 μg/ml) and IL-15 (150 IU/ml). Cell lines of hematological malignancies: RPMI-8226, H929, K562, RAJI, MOLM-13 and KG-1a were cultured according to the manufacturer‘s recommendations. ATRA treatment was applied to the cancer cells at 0, 1, 2.5, 5 and 10 μM concentration and CD1d expression was measured at 48 h and 72 h by flow cytometry. iNKT cells were co-cultured with cell lines pretreated with ATRA, α-GalCer or with ATRA in combination with α-GalCer. After 24 h of co-culture the percentage of dead cancer cells was measured by flow cytometry.
Results & Discussion: ATRA increased CD1d expression with the highest induction at 5 μM after 72 h in MOLM-13 (6 846 control vs. 162 397 treated median of fluorescence intensity (MFI)), RPMI-8226 (4 070 control vs. 23 869 treated MFI) and H929 (5 196 control vs. 120 946 treated MFI). In MOLM-13 the pre-treatment with ATRA only slightly increased the iNKTs killing ability (mean control 6%, mean treated 14.4%, p≤0.05) but, the addition of α-GalCer enhanced it significantly (mean dead cells 58.9%, p≤0.01). In RPMI-8226 and H929, the ATRA pre-treatment alone significantly improved the iNKT killing activity (RPMI-8226: mean control 12.3%, mean treated 68.3%; H929: mean control 33.7%, mean treated 66.1%; p≤0.01), and combination with α-GalCer improved it further (by 12% in RMPI-8226 and by 22% in H929; p≤0.05).
Conclusion: Our findings demonstrate that ATRA can upregulate CD1d expression in acute myeloid leukemia (MOLM-13) and multiple myeloma cell lines (RPMI-8226 and H929), which improves the cytotoxic activity of iNKTs but depends on the presence of lipid antigen.
Funding: Supported by the Ministry of Health of the Czech Republic project no. NW24-03-00079 and FNPI, 00669806.
Study program: Doctoral study – Anatomy, Histology and Embryology | Year of study: 2
ID: 1082
Tumor-Stroma Crosstalk in Urothelial Cancer | Martina Dolejšová
Tumor-Stroma Interactions Promote Stemness in Urothelial Carcinoma: Insights from Side Population Analysis
Authors: Martina Dolejšová (1), Michaela Kripnerová (2), Barbora Vítovcová (2), Martin Pešta (2),
Jiří Hatina (2)
Supervisor: Michaela Kripnerová (2)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (2) Department of Biology,
Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Cancer stem cells are heterogenous population of cells, which are thought
to be particularly responsible for insufficient treatment response, metastasis of tumours and
evasion of the immune response. Although they can differ even within a single tumour, they can
be defined by some common features. Using the flow cytometry method, we can demonstrate
the expression of surface antigens common to the immature cells, along with tumour specific
markers. Flow cytometry also allows the detection of cells with a hight efflux transporter activity.
These cells not only secrete chemotherapeutics, but also DNA-binding dyes, which can be
used in the “Side population analysis”, where cells with active transporters form a population
with a lower fluorescent signal, the so-called side population.
Objective: The aim of this study was to conduct a deeper analysis of the influence of tumour stroma on the stem phenotype of urothelial carcinoma using a methodology that I learned during my postgraduate internship in Innsbruck.
Material and Methods: To mimic the natural interaction between cancer and the stromal cells,
we used a proven co-culture system of the urothelial cancer cell line RT112, and cancer associate
fibroblast cell line BC44Fibr. The RT112 cancer cell line was seeded onto culture flasks containing
settled fibroblasts to achieve the best possible cell-to-cell contact. After six days of co-culture,
the proportion of side population cells were analysed using Vybrant™ DyeCycle™ Violet staining
(Thermo Fisher Scientific, USA) and to distinguish between the two types of cells we used CD 90
antibody. Cell suspension was subsequent analysed by flow cytometry (BD, USA). Obtained data
were compared to the result from RT112 monoculture.
Results & Discussion: We established an optimal seeding density for both cell types to maximize
cell interaction. Our 2D co-culture model not only allows the exchange of signalling molecules
between cells but also provides the possibility of communication via cell-to-cell contact. To distinguish
between the two cell types, the CD90 antibody was used, which specifically binds to
fibroblasts and not to RT112. The results of our pilot co-culture study suggest that the presence of
stromal fibroblasts can have a dramatic effect on the representation of cancer stem cells (CSCs).
The higher representation of cells with an active efflux pump in the tumour cell population in
direct contact with the stroma is consistent with our previously presented results in this cell line.
Conclusion: We successfully optimized a 2D co-culture model of the fibroblast line BC44Fibr
and urothelial carcinoma tumour cells. The effect of stromal cells, in accordance with our previous
results, increased the proportion of cells capable of actively secreting DNA-binding dye, i.e., cancer stem cells.
Funding: SVV-2024-260654, SVV-2025-260773, Academic Minigrant 4EU+ MA/25/F1/0/018, The
League Against Cancer Prague, The research internship was supported by the Erasmus.
Study program: Doctoral study – Medical Biology and Genetics | Year of study: 4
ID: 1142